cera such as Diaphanosoma brachyurum, Cerio- daphnia quadrangula, Daphnia cucullata, and. Daphnia magna as high-efficiency bacterium feeders according ...
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Apr. 1983, 0099-2240/83/041242-05$02.00/0 Copyright C 1983, American Society for Microbiology
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Vol. 45, No. 4
Ability of Daphnia Cell-Free Extract to Damage Escherichia coli Cells ORA HADAS,1 YEHUDA KOTT,2 URIEL BACHRACH,3 AND BENZION CAVARI1* Kinneret Limnological Laboratory, Tiberias, 14-102 Israel,1 The Technion, Israel Institute of Technology, Haifa, 32000 Israel,2 and Department of Molecular Biology, Hadassa Medical School, The Hebrew University, Jerusalem, 91000 Israel3
Received 20 August 1982/Accepted 27 December 1982
A cell-free extract of Daphnia magna was found to lyse Escherichia coli cells as shown by leakage of the enzymes alkaline phosphatase and 3-galactosidase from the bacteria. The cell-free extract was separated on Sephadex G-200, and the fractions showing an ability to lyse E. coli cells were isolated. The factor which was responsible for the lysis of the bacterial cells was probably a protein with a molecular weight of several thousands. Mg2' and Ca2+ ions augmented the activity of the Daphnia extract on E. coli cells.
Bacteria have been shown to be an important food source for Daphnia spp. in nature (3, 23). Peterson et al. (14) showed that Daphnia spp. can feed on small, natural bacterial flora in Lake Toolik, Alaska. Geller and Moller (W. Geller and H. Moller, Abstr. S.I.L. Congress. Kyoto, Japan, 1980, p. 221) grouped planktonic Cladocera such as Diaphanosoma brachyurum, Ceriodaphnia quadrangula, Daphnia cucullata, and Daphnia magna as high-efficiency bacterium feeders according to the mesh size of their filtering apparatus and suggested that the species composition of filter-feeding zooplankton is strongly influenced by the amount of suspended bacteria available as food. Bacteria are also important in the diet of other crustaceans, such as Tigriopus californicus (17), Tisbe holothuriae (19), Paramphiascella vararensis (19), and Tigriopus japonicus (7). Little is known about the biochemical and physiological digestion processes of bacteria by Daphnia spp. Quaglia et al. (18) found in the midgut of Daphnia spp. multivesicular-like bodies probably playing a role similar to that of lysozomes. They found that food was absorbed in a digested form. Radioactive carbon of algal origin was shown to participate in the biosynthesis of protein, lipids, and carbohydrates in Tigriopus and Calanus spp. (10) and can be seen in animal tissues after 24 h, as was shown for the copepod Temora longicornis (20). In this study we demonstrated the ability of a D. magna cell-free extract (CFE) to lyse Escherichia coli cells. MATERIALS AND METHODS Daphnia cultures. Daphnids were grown in flasks containing tap water. Ground fowl feces combusted at
200°C for 4 h were used as a food source. The regime for growing cultures consisted of 14 h of light and 10 h of dark at 20 to 22°C. Bacteria. E. coli cells isolated from the coliform community of an oxidation pond were used as a food source for Daphnia spp. E. coli cells were grown on M-9 medium (1) at 37°C without shaking; during the logarithmic phase, bacteria were centrifuged, washed twice, and suspended in filtered lake water (0.45-,umpore filters). This mixture served as the E. coli suspension. When induction of ,B-galactosidase was required, glucose was replaced by lactose in the growth medium.
Daphnia CFE preparation. About 100 daphnids were homogenized (in 3.0 ml of 3% NaCl) with an Ultra Turrax or Potter-Elvehjem homogenizer. The homogenate was centrifuged for 10 min at 9,000 x g (centrifuge from Measuring & Scientific Equipment, Ltd.), and the supernatant was used as the CFE. The effect of this extract on E. coli was examined as follows. CFE (0.2 ml) was incubated for 24 h with 0.2 ml of the bacterial suspension (109 cells ml-') and 2.6 ml of distilled water. Controls of only E. coli or CFE were used, and values for the controls were subtracted from experimental values. Lysis of E. coli was followed by a decrease in the absorbance at 540 nm (A540) and by the appearance of bacterial alkaline phosphatase activity in the supematant after the sample was centrifuged (20 min, 9,000 x g). CFE dialysate was prepared by dialyzing CFE against 1,000 volumes of water for 48 h, with one replacement of the water after 24 h. Four parallel experiments were run in duplicate, and variation among the CFEs was found to be not more than 10%o. CFE fractionation. CFE was prepared as described above from a large number of daphnids (ca. 3 x 103). The supernatant was applied to a Sephadex G-200 column (1.5 by 30 cm; Pharmacia Fine Chemicals). Elution was done with 0.85% NaCl, and 38 to 40 fractions (2 ml) were collected with an automatic microfractionator (Gilson Medical Electronics, Inc.,
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TABLE 1. Changes in cell turbidity and release of alkaline phosphatase from E. coli cells by Daphnia CFE phosphatase Reaction mixture A5,0 Alkaline 3 hactivity (A410) CFE + bacteria 0.48 0.39 Bacteria only 0.60 0.14 CFE + bacteria + Mg2+ 0.42 0.59 Bacteria + Mg2" 0.50 0.27 CFE + bacteria + Ca2` 0.42 0.42 Bacteria + Ca2" 0.44 0.14 CFE + bacteria + EDTA 0.42 a Bacteria + EDTA 0.68 a The enzyme was inhibited by EDTA.
Middleton, Wis.). The fractions
were
monitored for
A2,, and for alkaline phosphatase and P-galactosidase
activities. The lysing ability of Daphnia spp. was checked as follows. A 1.5-ml portion of each fraction was incubated for 48 h at 37°C with E. coli cells (109 cells mln') grown on lactose and then was centrifuged; the supernatant of each fraction was checked for alkaline phosphatase and ,-galactosidase activities. Controls of intact E. coli cells and elution solution were simultaneously run; values for the controls were subtracted from experimental values. Protein was determined in accordance with the method of Lowry et al. (12). Alkaline phosphatase acdvIty. The reaction mixture contained 0.2 ml of sample (fractions or supernatant), 2.8 ml of 0.05 M Tris buffer (pH 9.0), and 0.1 ml of Pnitrophenyl disodium orthophosphate (3.7 mg ml-'; BDH Chemicals Ltd., Poole, England). Enzymatic activity was expressed as the A410 after incubation at 37°C for 3 h. In the dialysate experiment incubation was done for 1.5 h only. ,B-Galactosidase activity. The reaction mixture contained 0.2 ml of sample, 1.5 ml of 0.2 M phosphate buffer (pH 7.5), and 0.4 ml of 0.01 M o-nitrophenyl-,BD-galactopyranoside (Sigma Chemical Co.) in 0.2 M phosphate buffer (pH 7.5). Enzymatic activity was expressed as the A420 after incubation at 28°C for 3 h. Gel electrophoresis. Samples of CFE (1.92 mg of protein) and fractions eluted from the Sephadex G-200 column which showed high alkaline phosphatase activity (before or after incubation with E. colt) were applied to polyacrylamide gels and tested for the origin of the enzyme (Daphnia spp. or E. coli). Three of the fractions which were suspected of causing the release of E. coli alkaline phosphatase were pooled together and dialyzed against water for 48 h. The dialysis bag was then placed in sucrose for 1 h. The small volume which remained in the bag and other fractions which showed alkaline phosphatase activity (of Daphnia origin) were mixed with 50o sucrose and 1 drop of bromophenol blue, which served as a marker. Each sample was loaded on the gels, and electrophoresis was continued for 45 to 60 min at 1.5 mA (for each tube). Gels were stained for alkaline phosphatase with naphthyl acid phosphate medium (1 mg ml-1; Mann Research Laboratories) and fast blue RR salt (1 mg ml-1; Sigma) until color development. Preparation of gels was done in accordance with the method of Wynne (24).
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RESULTS Daphnia CFE was tested for its ability to lyse E. coli cells. As an indicator of lysis, the release of alkaline phosphatase into the medium was checked. When CFE was incubated with E. coli cells, a greater release of alkaline phosphatase into the medium was observed: A410 with Daphnia CFE, 0.39; A410 without Daphnia CFE, 0.14 (Table 1). In addition, when the A540 was checked as an estimation of cell density a decrease in the absorbance was noted. When E. coli cells were incubated with the CFE for 24 h, the A540 was 0.48; the A540 was 0.60 when E. coli cells were incubated without the CFE. The addition of 10-3 M EDTA, 10-3 M Mg2+, or 10-3 M Ca2+ to the incubation mixture enhanced the activity of CFE on E. coli cells (Tables 1 and 2). The above results were obtained when incubation was done at 27°C; similar results were obtained when incubation was carried out at 37°C. When boiled CFE was added to the bacterial suspension, less leakage of alkaline phosphatase was noted: A410 with boiled CFE, 0.27; A410 with unboiled CFE, 0.39. In the absence of CFE the A410 obtained was 0.18. These results may imply the protein nature of the factor in the CFE that causes damage to E. coli cells. When Mg2+ (10-3 M) was added to the reaction mixture, the effect was even greater: A410 with unboiled CFE, 0.59; A410 with boiled CFE, 0.20. The same set of experiments was also run with a dialysate of CFE, and the results were compared with those for CFE (Table 2). Increasing amounts of CFE or dialysate resulted in a greater amount of alkaline phosphatase being released. When the amount of CFE was doubled, the A410 increased from 0.29 to 0.82; when the amount of dialysate was tripled, the A410 increased from 0.21 to 0.75. CFE was loaded on a Sephadex G-200 column and eluted with 0.85% NaCl, and 2-ml fractions were collected. Subsamples of each fraction were checked for alkaline phosphatase and ITABLE 2. Effect of Daphnia dialysate and CFE on E. coli cell lysis and release of alkaline phosphatase Alkaline Reaction mixture
Aw3
phosphatase activity (A410) 1.5 h-1
CFE + bacteria
Bacteria only CFE + bacteria + Mg2` Bacteria + Mg2" Dialysate + bacteria Bacteria only Dialysate + bacteria + Mg2" CFE (double amount) Dialysate (triple amount)
0.3 0.67 0.26 0.41 0.53 0.59 0.39 0.29 0.21
0.27 0.18 0.40 0.18 0.23 0.18 0.38 0.82 0.75
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HADAS ET AL.
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U
Se
Fraction No.
I =J
lEy
--I Fraction
0
No.
FIG. 1. Alkaline phosphatase activity in fractions of Daphnia CFE before ----) and after incubation with E. coli cells. CFE (2.4 ml, 1.92 mg ml-1 of protein) was loaded on the column. The magnification of fractions 15 through 30 is given in the insert.
galactosidase activities. A 1.5-ml amount of each fraction was incubated with E. coli cells for 24 and 48 h and then checked again for alkaline phosphatase and P-galactosidase activities. Most of the enzymatic activities (alkaline phosphatase and P-galactosidase) appeared in fractions 6 through 15 (Fig. 1 and 2). Another peak of alkaline phosphatase activity appeared in fractions 18 through 21 only when these fractions were incubated with E. coli cells (Fig. 1). Several peaks of P-galactosidase activity in fractions 18 through 34 appeared after incubation of these fractions with E. coli cells (Fig. 2). The origin of the alkaline phosphatase was studied by subjecting the fractions containing the alkaline phosphatase activity to polyacrylamide gel electrophoresis. A sample from fraction 10 before and after incubation with E. coli cells was compared with a sample from fractions 19 through 21 after incubation with E. coli cells. From Fig. 3 it is clear that the alkaline phosphatase in fraction 10 was different from the alkaline phosphatase in fractions 19 through 21. The appearance of fraction 10 in the polyacrylamide gel was similar before and after incubation with E. coli cells and similar to that in the Daphnia CFE, indicating that the enzyme in fraction 10 originated from the Daphnia spp. Fractions 19 through 21, on the other hand, contained alka-
line phosphatase activity only after incubation with E. coli cells, and the enzyme in fractions 19 through 21 showed a running pattern different from that of the enzyme in fraction 10. The running pattern of the enzyme in fractions 19 through 21 was similar to that of the commercial alkaline phosphatase prepared from E. coli cells (Fig. 3); it may indicate the E. coli origin of the enzyme in fractions 19 through 21. DISCUSSION Studies dealing with the ingestion of algae and bacteria by Daphnia spp. referred mostly to the intake processes of different kinds of algae (2, 8, 15, 16, 21) or bacteria (4, 5, 11, 13, 22). Others determined the feeding rate or the filtering rate of Daphnia spp. grown on these food sources (9, 13). In natural aquatic ecosystems bacteria play an important role as a food source for Daphnia spp. (3, 23) and other crustaceans (19). In previous studies (6) we showed that Daphnia spp. ingest bacteria and algae. The purpose of the present study was to investigate the ability of a Daphnia CFE to lyse E. coli cells. The criterion for the lysis of E. coli cells by Daphnia extracts was the leakage of the enzymes alkaline phosphatase and P-galactosidase from the cells. These enzymes are located in the periplast of the bacterial cell, so, after damage to the bacterial
VOL. 45, 1983
LYSIS OF E. COLI BY DAPHNIA CELL-FREE EXTRACT
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owI*
2.0 20
25 Froction No.
D
Fraction No.
) incubation FIG. 2. P-Galactosidase activity in fractions of Daphnia CFE before (- - - -) and after ( with E. coli cells. CFE (2.4 ml, 1.92 mg mlP 1 of protein) was loaded on the column. The magnification offractions 17 through 30 is given in the insert.
FIG. 3. Gel electrophoresis of CFE and offractions before and after incubation with E. coli cells. (A) 50 ,ul of CFE; (B) 50 Il1 of fraction 10 after incubation with E. coli cells; (C) 30 p.l of fraction 10 before incubation with E. coli cells; (D and E) 100 p.l of pooled fractions 19 through 21 after incubation with E. coli cells; (F) 10 pl of commercial E. coli alkaline phosphatase (Sigma).
cell wall, these enzymes are immediately released into the medium. Another parameter was the decrease in the turbidity of the bacterial culture caused by the lysis of the bacteria. The addition ofDaphnia CFE to E. coli cells resulted in a much greater release of enzymes from the E. coli cells than from controls without CFE. Mg2+ or Ca2+ ions or the chelating agent EDTA enhanced this process when added to the reaction mixture. This enhancement may have been due to the action of these agents on the cell wall (EDTA) or to the assistance given by these agents to the digesting factor present in the Daphnia CFE. The protein nature of the factor was demonstrated by the decreased lysis caused when boiled CFE was added to E. coli cells. The digesting factor was retained in the dialysis bag (Table 2), suggesting that its molecular weight is greater than 10,000. When Daphnia CFE was separated on a Sephadex G-200 column and each fraction was incubated with E. coli cells, bacterial enzymes (alkaline phosphatase and P-galactosidase) was found connected only with fractions 19 through 25. No such enzymatic activity was connected with these fractions before incubation with E. coli cells (Fig. 1 and 2). Since Daphnia spp. and microflora connected with Daphnia spp. also contain alkaline phosphatase, the origin of enzymes released was checked by poly-
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HADAS ET AL.
acrylamide gel electrophoresis. Fractions 19 through 21 were found to have the same band pattern as that of a commercial E. coli alkaline phosphatase, indicating the bacterial origin of the enzyme in these fractions. Fraction 10 showed a completely different running pattern, which did not change before and after incubation with E. coli cells, suggesting that the enzyme in this fraction was of Daphnia origin. The damage caused to the E. coli cells by the Daphnia CFE, as represented in the release of the enzymes, is probably the first step in the breakdown of E. coli cells by Daphnia spp. Studies are now under way to examine the digestion mechanism in intact Daphnia spp.
APPL. ENVIRON. MICROBIOL.
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11. 12. 13. 14. 15. 16.
1. 2.
3.
4. 5.
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7.
8. 9.
LITERATURE CITED Adams, M. H. 1959. Bacteriophages, p. 446. Interscience Publishers, Inc., New York. Arnold, D. E. 1971. Ingestion, assimilation, survival and reproduction by Daphnia pulex fed seven species of bluegreen algae. Limnol. Oceanogr. 16:906-920. Coveney, M. F., G. Cronberg, M. Enel, K. Larsson, and L. Olotssof. 1978. Phytoplankton, zooplankton and bacteria standing crop and reproduction relationship in eutrophic lake. Oikos 29:5-21. Gophen, M. 1977. Feeding of Daphnia on Chlamydomonas and Chlorobium. Nature (London) 265:271-272. Gophen, M., B. Z. Cavari, and T. Berman. 1974. Zooplankton feeding on differentially labelled algae and bacteria. Nature (London) 247:393-394. Hadas, O., B. Z. Cavari, Y. Kott, and U. Bachrach. 1982. Preferential feeding behaviour of Daphnia magna. Hydrobiblogia 89:49-52. Hanaoka, H. 1973. Cultivation of three species of pelagic microcrustacean plankton. Bull. Plankton Soc. Jpn. 20:19-29. Kerstg, K., and W. Hoterman. 1973. The feeding behaviour of Daphnia magna studied with the coulter counter. Verh. Int. Ver. Limnol. 18:1435-1440. Kersting, K., and V. Leeuv. 1976. The use of the coulter
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counter for measuring the feeding rates of Daphnia magna. Hydrobiologia 49:233-237. Khaylov, K. M., and V. Y. Yerokbin. 1971. Utilization of dissolved organic matter by the crustaceans Tigriopus bravicornis and Calanus finmerchicus. Oceanology 11:95-103. Lampert, W. 1974. A method for determining food selection by zooplankton. Limnol. Oceanogr. 19:995-998. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265-275. McMahon, J. W., and F. H. Rigler. 1965. Feeding rate of Daphnia magna (Straus) in different foods labelled with radioactive phosphorus. Limnol. Oceanogr. 10:105-115. Peterson, B. J., Y. E. Hobbie, and J. F. Haney. 1978. Daphnia grazing on natural bacteria. Limnol. Oceanogr. 23:1039-1044. Porter, K. G. 1973. Selective grazing and differential digestion of algae by zooplankton. Nature (London) 244:179-180. Porter, K. G. 1975. Viable gut passage of gelatinous green algae ingested by Daphnia. Verh. Int. Verein. Limnol. 19:2840-2850. Provasoll, J., K. Shiraishi, and J. R. Lance. Nutritional idiosyncrasies of Artemia and Tigriopus monoxenic culture. Ann. N.Y. Acad. Sci. 77:250-261. Quaglia, A., B. Sabelli, and L. Villani. 1976. Studies on the intestine of Daphnidae (Crustacea, Cladocera): ultrastructure of the midgut of Daphnia magna and Daphnia obtusa. J. Morphol. 150:711-726. Rieper, M. 1978. Bacteria as food for marine harpacticoid copepods. Mar. Biol. 45:337-345. Smith, S. L., and B. K. Hall. 1980. Transfer of radioactive carbon within the copepod Temora longicornis. Mar. Biol. 55:277-286. Taub, F. B., and A. M. Dollar. 1968. The nutritional inadequacy of Chlorella and Chlamydomonas as food for Daphnia pulex. Limnol. Oceanogr. 13:607-617. Tezuka, Y. 1971. Feeding of Daphnia on plankton bacteria. Jpn. J. Ecol. 21:121-134. Weglenska, T. 1971. The influence of various concentradons of natural food on the development, fecundity and production of planktonic crustacean ffiltrators. Ekol. Pol. 30:427-447. Wynne, D. 1977. Alterations in activity of phosphatase during the Peridinium bloom in Lake Kinneret. Physiol. Plant. 40:219-224.
